This is a revised application to comprehensively examine the roles of hantavirus nucleocapsid protein (N) in virus replication. Hantaviruses include Category A pathogens and are members of the Bunyaviridae family which includes both Category A and C pathogens. It is likely that the principles of RNA packing that take place during hantavirus infection will be applicable to the other members of the hantavirus genus the entire Bunyaviridae family, and other medically important segmented minus strand RNA viruses such as influenza. All minus strand, segmented RNA viruses have genome segments that are found in "panhandle" formation via interaction of the genome termini. We have found that this viral RNA (vRNA) panhandle is the primary high affinity binding substrate for hantavirus N. We will carry out complementary in vitro and in vivo experiments to characterize the determinants required for correct packaging of viral RNA into particles. We have also found that hantavirus N protein is a robust RNA chaperone that transiently dissociates higher order RNA structure to facilitate formation of higher order structures with biological function. Thus, we will carry out experiments to characterize the role of N- associated RNA chaperone function in virus replication. In particular we will characterize the role of N in panhandle formation and in the initiation of viral RNA replication. We have also found that N mimics the activity of multiple cellular peptides of the cap-binding complex during translation initiation. We will further characterize the role of N in this process. Although N functions in multiple steps in replication, we think that common intrinsic activities of N mediate these biological functions. In addition to characterizing the features of the biology of hantavirus N, we will use our observed high affinity interaction between N and the vRNA panhandle to identify molecules that inhibit N function and virus replication. PUBLIC HEALTH RELEVANCE: Hantaviruses cause hantavirus pulmonary syndrome which has a high mortality rate in humans. Other viruses closely related to hantaviruses cause a diverse set of human diseases including hemorrhagic fever. The goal of our proposed work is to identify salient biological functions of one of the viral proteins crucial to virus replication and to identify drugs effective in blocking the replication of these viruses.